Chem. Res. Toxicol. 1993,6, 852-857
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Both Cytochromes P450 2E1 and 1Al Are Involved in the Metabolism of Chlorzoxazone V6ronique Carriere,? Thierry Goasduff,$ Damron Ratanasavanh,§ Fabrice Morel,ll Jean-Charles Gautier,? And& Guillouzo, I Philippe Beaune,? and FranCois Berthou*J
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Laboratoire de Biochimie, Facultk de Mkdecine, B.P. 815-29285, Brest, France, INSERM U- 75, Uniuersitk Renk Descartes, CHU-Necker, Paris, France, Laboratoire de Pharmacologie, Facultk de Mkdecine, Brest, France, and INSERM U-49, CHU-Pontchaillou, Rennes, France Received May 24,1999
Chlorzoxazone, a centrally acting muscle relaxant, was previously shown to be hydroxylated on carbon 6 specifically by cytochrome P450 2E1. Accordingly, this drug has been proposed as a potential noninvasive in-vivo probe for screening the hepatic P450 2E1 activity. This study was carried out to test the specificity of such a substrate when first experiments conducted by using human hepatocyte cultures showed that the chlorzoxazone 6-hydroxylation activity increased after 3-methylcholanthrene treatment of cells. Indeed, the ability of both rat and human hepatocytes to metabolize chlorzoxazone significantly increased after treatments by 3-methylcholanthrene alone or plus ethanol, suggesting the involvement of P450 1A enzymes in this oxidative reaction. Identical results were obtained by in-vivo treatment of rats with four inducers of P450 1A enzymes, namely, 0-naphthoflavone, isosafrole, Arochlor 1254, and 3-methylcholanthrene. Furthermore, the chlorzoxazone 6-hydroxylation activity was inhibited by both a-naphthoflavone and dimethyl sulfoxide, both known to inhibit P450 1A and P450 2E1 activities, respectively. Finally, the use of yeasts genetically engineered for expression of human P450 1AlI1A2, 2C9, and 3A4 demonstrated that P450 1Al was significantly involved in this catalytic activity. In conclusion, these results taken together suggest that chlorzoxazone should be used with precaution as in-vivo tool for evaluating P450 2E1. However, the relative K, of P450 1Al and 2E1 for chlorzoxazone and, on the other hand, the relative levels of these two enzymes in the human liver suggest that P450 2E1 would generally be the major form metabolizing chlorzoxazone in-vivo.
Introduction Cytochrome P450 comprises a superfamily of hemethiolate protein isozymes involved in the oxidative metabolism of a large array of endogenous and exogenous compounds, including drugs, environmental pollutants, steroids, prostanglandins, and fatty acids (2).Isozymes are generally characterized by a specific substrate that they metabolize stereospecifically (3,4).Recently, chlora compound zoxazone [5-chloro-2(3H)-benzoxazolonel, used therapeutically as a centrally acting muscle relaxant, was shown to be hydroxylated on carbon 6 specifically by cytochrome P450 2E1 (P4502E1)1(5). This enzyme is of considerable relevance to human health, first because it is inducible by acute and chronic alcohol ingestion, diabetes, fasting, pyrazole, acetone, and isoniazid (6) and second because it is involved in the oxidation of toxic and To whom correspondence should be addressed. Fax (33)98 31 64 74. t INSERM U-75, Universit.6 R e d Deascartes. t Laboratoire de Biochimie, Facult.4 de MBdecine. t Laboratoire de Pharmacologie, Facult4 de MBdecine. I INSERM U-49. 0 Abstract published in Aduance ACS Abstracts, October 1, 1993. 1 Abbreviations: 3-MC, 3-methylcholanthrene; PBS, (potassium) phosphate buffer saline = 0.01 M potassium phosphate buffer, pH 7.4, 0.138 M NaC1,2.7 mM KCI; &NF, 8-naphthoflavone or 5,6-naphthoflavone; a-NF, a-naphthoflavone or 7,&benzoflavone; MROD, 7-methoxyreaorufin Odemethylase; EROD,7-ethoxyreeorufinO-deethyh,M&O, dimethyl sulfoxide; PCR, polymerase chain reaction; cytochrome P450 2E1, P450 2E1 (EC 1.14.14.1). The updated recommended nomenclature for P460 enzymesis according to Nelson et al. (I).The name 'cytochrome" has been abandoned according to the Nomenclature Committee of the InternationalUnionof Biochemistrynomenclatureof the electron-transfer proteins, the appropriate name being 'heme-thiolate protein".
many carcinogenic chemicals (7, 8). So accordingly, chlorzoxazone has been proposed as a potential noninvasive in-vivo probe for screening the liver P450 2E1 activity (4, 8). Given the likely broad application of the chlorzoxazone 6-hydroxylationfor in-vivo and in-vitro tests of P450 2E1 activity, specificity of this substrate was reevaluated. This study was carried out using cDNA expression, rat and human hepatocyte cultures treated with ethanol and/or 3-methylcholanthrene,and in-vivotreatments of rats with pyrazole, acetone, and different inducers of P450 1A enzymes. The results have highlighted the overlapping specificity of chlorzoxazone. These experiments clearly demonstratedthat chlorzoxazone is not a specific substrate of P450 2E1 and importantly can be metabolized by P450 1Al.
Materials and Methods Chemicals. All reagents were of analytical grade. Chlorzoxazone [5-chloro-2(3H)-benzoxazolonelwas purchased from Sigma Chemical Co. (StLouis,MO) and ita 6-hydroxy metabolite was a gift from Dr. Peter (University of Erlangen-Niirnberg, Germany). Solvents were of HPLC grade from Merck (Darmstadt, Germany). 0-Naphthoflavone (0-NF or 5,6-benzoflavone), a-naphthoflavone (a-NF or 7,&benzoflavone), isosafrole, and 3-methylcholanthrene (3-MC) were from Sigma. Arochlor 1254 was from Chem-Services (Interchim, Montlupn, France). Rat Treatment and Preparation of Microsomes. Male Wistar rats (180-200 g from IFFA-CREDO, L'Arbresle, France) were maintained on a 12-h light/dark cycle in a temperaturecontrolled environment. They had a free access to water diet
0893-228~/93/ 2706-0852$04.O0/0 0 1993 American Chemical Society
Chlorzoxazone Is a Substrate of P450 2El and 1Al and standard food (M-25 Biscuit Extralabo, from Pihtrement, Provins, France). They were killed when 6 weeks old. Acetone was administered intragastrically a t 1and 2 mL/kg of a 25% acetone aqueous solution, 42 and 18 h, respectively, before killing. Pyrazole was injected intraperitoneally a t 200 mg/kg/day for 3 days before sacrifice. 3-MC, isosafrole, B-NF, and Arochlor 1254were injected intraperitoneally in solutions of 1% ,l o % , 1% ,and 20% (w/v) in corn oil, respectively, a t dosages of 40 mg/kg/day for 3 days, 200 mg/kg/day for 3 days, 80 mg/ kg/day for 3 days, and 500 mg/kg for 1 day before sacrifice, respectively. Animals were starved for 12 h before the sacrifice. Liver microsomes were prepared as described previously (9)and stored a t -80OC until use. H e p a t o c y t e C u l t u r e s a n d Cell T r e a t m e n t . (A) R a t Hepatocytes. Hepatocytes were isolated from male Wistar rats (200-250 g) by collagenase perfusion of the whole liver as previously described (10,111.Cells were seeded a t a density of 2.5 X lo6per 28-cmZPetri dish in 4 mL of nutrient medium. The culture medium consisted of a mixture (3/1 v/v) of minimum essential medium and medium 199, added with 10 pg/mL bovine insulin, 0.2% bovine serum albumin, and 10% fetal calf serum. Cells were treated with 90 mM ethanol, 5 pM 3-MC in MezSO, and 10 mM pyrazole in PBS or 5 pM 3-MC 90 mM ethanol during 3 days. The culture medium containing inducers was renewed every day. Control cells were treated with the same final concentration of MezSO (0.5% v/v). After cell treatment, chlorzoxazone a t 0.2 mM was incubated for 24 h in the absence of inducers. The medium was removed, treated with 150 pL of 43 % H a 0 4 , and extracted with 6 mL of chloroform/2-isopropanol (85/15 v/v). The organic phase was dried by filtration on sodium sulfate and then evaporated to dryness under a nitrogen stream and redissolved in 200 pL of HPLC mobile phase. HPLC was carried out as described below. (B) Human Hepatocytes. Adult human hepatocytes were prepared by the two-step collagenase perfusion method as previously described (10,11)from human livers obtained from two adult kidney transplantation donors (R053, male 38-yearold, and FR3, male 70-year-old) who died from traffic accidents. Sampling was made following the recommendationsof the French ethical committee. Dietary habits and exposure to environmental chemicals before death were not known. The cells were seeded a t a density of lo7 in 10 mL of nutrient medium containing 10 pg/mL bovine insulin, 0.2% bovine serum albumin, and 10% fetal calf serum. The culture medium was renewed 24 h later without fetal calf serum but containing 90 mM ethanol or 5 pM 3-MC or 90 mM ethanol plus 5 pM 3-MC. The culture medium was renewed every day. After 72-h treatment of culture, cells were washed with chilled PBS and collected in 5 mL of PBS by scraping with a rubber policeman and centrifuged a t 100g. The pellets were stored at -80 "C until preparation of microsomal fractions. Cultures with or without 0.5% MezSO (v/v) were used for control for the same period of culture. Hepatocytes were disrupted with a glass-glass Potter-Elvehjem device in 0.1 M potassium phosphate buffer (pH 7.4) containing 0.25 M sucrose, 10 mM EDTA, and 0.1 mM dithiothreitol. Subcellular fractions were obtained by differential centrifugation. The final microsomal pellet after a second centrifugation a t 105000g was resuspended in 0.1 M potassium phosphate buffer (pH 7.4) with 1 mM dithiothreitol, 10 mM MgC12, and 20% glycerol (v/v). cDNA Expression of Different P450i n Yeasts. P450 1Al/ 1A2 and P450 2C9 coding sequences were cloned by the polymerase chain reaction (PCR) from human liver cDNA templates and were inserted in the yeast expression vector YeDP6O (12).Human P450 3A4 coding sequence was inserted in YeDP6O by gap repair (13). Each expression vector was introduced into the engineered Saccharomyces cerevisiae strain W(R) which overexpresses yeast NADPH-P450 reductase when grown with galactose as carbon source (14).Culture medium 55 (containing galactose) without adenine, spheroplast preparation by enzymatic digestion of yeast cell walls, and subcellular fractionation were as previously described (13, 14).
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Chem. Res. Toxicol., Vol. 6, No. 6,1993 853 Immunoblot Quantification of P450 1A a n d P450 2El. Microsomal proteins were separated by sodium dodecyl sulfatepolyacrylamide (9 % acrylamide) gel electrophoresis according to Laemmli (15). Proteins were then electrophoretically transferred to nitrocellulose sheets (Hybond-C, Amersham International, Amersham, England) according to Towbin et al. (16). Nitrocellulose sheets were blocked with PBS containing 3% (w/ v) bovine serum albumin, 10% fetal calf serum (Sigma), and 0.05% Tween 20 (v/v) for 30 min a t room temperature and incubated overnight with either anti-rat P450 2E1 antibody (Oxygene, Dallas,TX) or anti-rat P450 1A [prepared from rat induced by 8-NF according to Guengerich et al. (17)l. The nitrocellulose sheets were processed as previously described (18, 19). In outline, they were incubated with peroxidase-labeled anti-rabbit IgG or anti-rabbit IgG and horseradish peroxidase anti-peroxidase complex (ICN Biomedicals, Bucks, U.K.) raised against rabbit IgG. Peroxidase staining was developed with PBS containing 16% methanol, 2.8 mM chloronaphthol, and 0.8% (v/v) HzOz. The amounts of different P450s (P450 1Al/lA2 and P450 2E1) were determined by densitometry of the spots scannerized by a scanner (Scanjet IIC, Hewlett-Packard, Palo Alto,CA) and quantified by means of a microcomputer program (Scan Analysis, Biosoft, Cambridge, U.K.). Isolation of RNA, N o r t h e r n Blot Analysis, a n d P450 Probes. Total RNA was extracted from cultured hepatocytes by the guanidium thiocyanate/CsCl method of Chugwin et al. (20).Cells were washed with chilled PBS and scraped and lysed in a solution containing 0.1 M sodium acetate (pH 5.5), 5 M guanidium thiocyanate, 1 mM EDTA, 2% (w/v) sarcosyl, and 5 % (v/v) 2-mercaptoethanol. Homogenate5were centrifuged at 12000g for 15 min. Supernatants were then centrifuged on a 5.7 M CsCl cushion a t 85000g for 22 h. RNA pellets were washed, dried, and dissolved in sterile water and stored a t -80 OC. RNA aliquots (10 pg) were subjected to electrophoresis in a denaturing formaldehyde agarose gel and transferred onto Hybond-N+ sheets (Amersham International, Amersham, England). The sheets were prehybridized and successively hybridized with several [82PlcDNAprobes: P450 1A1, P450 1A2, and P450 2E1 cDNA obtained by reverse transcription, then PCR amplification from adult human liver mRNA (12). Relative amounts of mRNA were determined by scanning the developed X-ray films as described for proteins. Monooxygenase Activities. Microsomal protein contents were determined using the Bradford method according to the procedure recommended by the supplier (Bio-rad, Munich, Germany). Total P450 concentrations were determined by Fez+ - CO versus Fez+difference spectrophotometry as described by Omura and Sato (21)with a molecular extinction coefficient of 91 mM-l-cm-l. 7-Methoxyresorufin 0-demethylase (MROD) and ethoxyresorufin 0-deethylase (EROD) activities were determined by spectrofluorimetry according to the method of Prough et al. (22). Substrates (Boehringer, Mannheim, Germany) were added in 10 pL of MezSO solution to 2 mL of 0.1 M potassium phosphate buffer (pH 7.4) to obtain a final concentration of 5 pM. The protein concentration was 0.075 mg/mL. The reaction was initiated by addition of 0.5 mM NADPH. Fluorescence was directly measured in cuvettes under stirring for 2 min. Calibration was made by addition of 53 pmol of resorufin to the reaction medium. The 6-hydroxylation of chlorzoxazone in microsomal preparations of rat liver was measured according to Peter et al. (5), except that 1 mM NADPH was used instead of a NADPHgenerating system. Substrate was added to the incubation mixture a t a concentration of 0.4 mM. Microsomal proteins, 0.4 mg/mL, were incubated for 20 min at 37 OC. Extraction was performed by 6 volumes of a chloroform/2-propano1(85/15v/v) mixture after addition of 50 pL of 20% H3P04 The organic phase was reduced to dryness under a stream of nitrogen at 60 OC; 100 pL of acetonitrile/water (75/25 v/v) was added, and 50 pL was injected into HPLC. When microsomal samples from yeasts genetically enginereed were tested, 2 mg of protein was
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Chem. Res. Toxicol., Vol. 6, No. 6, 1993
Table I. Effect of Different Treatments of Human Hepatocyte Cultures on mRNAs and P450 Expression of P450 1A1, P450 1A2, P450 2E1, and Chlorzoxazone 6-Hydroxylation treatment control Me2SOc EtOHd 3-MC" 3-MC + EtOH"
P450 1 A l mRNAa 1 1.1
1.8 226 86
P450 1A2 mRNAa
P450 2E1 mRNAa
P450 1Al/lA2 proteinsa
1
1
1
1
1
0.6 0.8 250 101
3.7 0.8 1.4 1.2
1.8
7.3 7.7 7 3.9
1.2 2.7 3.6 5.3
1.2
13.4 12.3
P450 2E1 proteina
chlorzoxazone 6-hydroxylase*
a Results are the values of 1experiment conducted with liver R053. Results are the means of 2 human livers, each with three dishes. Control chlorzoxazone 6-hydroxylation was 15 pmol/(min*mgof protein) at t = 72 h while this activity at t = 0 h was 52 f 16 (n = 3; range 37-69) pmol/(min-mgof protein). Me2SO: 0.5% (v/v), Le., 73 mM. 90 mM EtOH was dissolved into culture medium. e 3-MC (5 pM) and 3-MC EtOH (5 p M + 90 mM), respectively, were dissolved in 0.5% (v/v) Me2SO.
+
incubated for 60 min a t 37 "C with variable concentrations of chlorzoxazone (4-1200 pM) added in 2 p L of Me2SO. The turnover number was determined by incubating 1 mg of microsomal protein with 0.4 mM chlorzoxazone and l mM NADPH for 60 min a t 37 OC. In both experiments, metabolites were extracted as above described. Quantification of the 6-hydroxylated metabolite was carried out by HPLC on a Nucleosil C-18, particle diameter 5 pm, 250 X 4.6 mm column (Interchim MontluCon, France). The mobile phase a t a flow rate of 1mL/min consisted of water containing 0.5% (v/v) glacial acetic acid/acetonitrile (75/25 v/v) for 30 min and then linearily modified to 25/75 in 15min by means of pump SP 8700 (Spectra-Physics, San Jose, CA). UV detection was performed a t 287 nm (Spectromonitor 111,LDC, Riviera Beach, FL). Calibration curves were performed by injecting increasing amounts of 6-OH-chlorzoxazone. The linearity of 6-OH derivative production was checked by varying the protein concentration (0.5-2 mg) and incubation times (30-60 min). The identity of the 6-OH-chlorzoxazone peak was confirmed by its chromatographic behavior and UV and fluorescence (&-itation = 290 nm and L m h i o n = 326 nm) characteristics.
Results Human Hepatocyte Culture. When human hepatocyte cultures were treated with 90 mM ethanol for 3 days, the chlorzoxazone 6-hydroxylation increased 2.7-fold vs control hepatocytes cultured for the same period of time (Table I). Unexpectedly, this monooxygenase activity also increased vs control by 3.6- and 5.3-fold by 3-MC and 3-MC plus ethanol treatments, respectively. However, chlorzoxazone 6-hydroxylation activities after such treatments of human hepatocyte cultures increased vs control hepatocytes but not vs t = 0 time cultures (Table I). Therefore, the results presented in Table I show that chlorzoxazone6-hydroxylaseincreased not only by ethanol treatment but also by 3-MC and 3-MC + EtOH treatments. Indeed, EtOH was able to increase both P450 2E1 protein (7.7-fold vs control) and chlorzoxazone metabolism (2.7fold vs control) without significant modifications of P450 2E1 mRNA. The effect of Me2SO was more complex. Indeed, treatment with 73 mM Me2SO increased both P450 2E1 protein and mRNA (7.3- and 3.7-fold vs control, respectively) without significant modification of chlorzoxazone 6-hydroxylation. This result is consistent with the findings of Eliasson et al. (23)who demonstrated that P450 2E1 (P450j)increased about 4-fold in rat hepatocyte cultures treated with 50 mM Me2SO. Simultaneously, Me2SO was shown to be a competitive inhibitor of P450 2E1 monooxygenase activities in rabbit microsomes [P4503d (24)] so that chlorzoxazone metabolism was not modified by Me2SO treatment. Therfore, the increase of of chlorzoxazone 6-hydroxylase (3.6- and 5.5-fold, respectively) observed with 3-MC and 3-MC + EtOH treatments could not be due to the P450 2E1 variations. Indeed, 73
8001
Control
MePO
3-MC
3-MC + EtOH EtOH
I'yrazolc
Treatment of culture
Figure 1. Effect of different treatments of rat hepatocyte cultures on chlorzoxazone 6-hydroxylation activity. Chlorzoxazone 6-hydroxylation activity was measured directly on cultures of rat hepatocytes incubated 24 h with 0.2 mM chlorzoxazone. Cells were previously treated with 0.5% (v/v) Me2SO,5 pM 3-MC, 5 pM 3-MC 90 mM ethanol, 90 mM ethanol, or 10 mM pyrazole during 3 days. Values represent averages f SD of 6 experiments, i.e., 2 rat livers each cultured in 3 dishes. Chlorzoxazone 6-hydroxylation activities measured a t t = 4 and 72 h were 320 f 36 and 160 i 12 pmo1/24 h/dish (n = 4 experiments), respectively.
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mM Me2SO was also added to these cultures and therefore inhibited this P450 2E1 activity. So, we can conclude that it was quite likely that the chlorzoxazone 6-hydroxylation increases were due to P450 1A enzymes induced by 3-MC treatments. The levels of total P450 1A (1Al + 1A2) proteins vs control were dramatically increased by 3-MC and 3-MC + EtOH treatments (13.4- and 12.3-fold vs control, respectively). Me2SO had only a slight effect on P450 1A proteins. As the used electrophoretic conditions could not separate with good resolution the human P450 1 A l and P450 1A2 proteins (contrary to rat orthologous P450 lA), it was not possible to know which protein of the P450 1A family was involved in chlorzoxazone metabolism. Indeed, the Northern blot technique demonstrated that the two mRNAs were dramatically increased by 3-MC and 3-MC + EtOH treatments (Table I) in correlation with chlorzoxazone metabolism. In order to confirm these results, complementary experiments were performed by using rat hepatocyte cultures, rat liver microsomes, and yeasts expressing various human P450s. R a t Hepatocyte Culture. Figure 1 shows the chlorzoxazone 6-hydroxylation activities (expressed as pmol/ 24 h/dish of 2.5 X lo6 cells) as a function of treatment of rat hepatocyte cultures. This monooxygenase activity was referenced against control cultures, i.e., cultured for the same period of time as treated cultures. During these 3
Chem. Res. Toxicol., Vol. 6, No. 6,1993 866
Chlorzoxazone Is a Substrate of P450 2E1 and 1Al Table 11. Fold Increase of Monooxygenase Activities in Microsomal Preparations of Rats Treated with Different Inducers. inducer
chlorzoxazone 6-hydroxylationb
MRODC
ERODd
acetone pyrazole Arochlor 1254 6-NF isosafrole 3-MC
2.5 (f0.25) 1.75 ( f O . l l ) 1.97 (f0.13) 2.07 (f0.47) 3.24 (f0.98) 3.02 (f0.43)
0.35 (f0.04) 0.38 (f0.02) 6.69 (f1.95) 6.50 (f2.51) 5.35 (f1.45) 3.40 (f1.50)
0.64 (f0.05) 0.80 (f0.16) 22.76 (f4.40) 29.24 (f2.36) 26.92 (f4.67) 26.1 (f5.50)
aValues are reported as the mean (fSD) of at least 3 rata. Abbreviations: @-NF,6-naphthoflavone; 3-MC, 3-methylcholanthrene; MROD, methoxyresorufin demethylase; EROD, ethoxyresorufiideethylase. Control activitywas 612 (f156) pmol/(min.mg); n = 4 rata. Control activity was 685 (45.5)pmol/(min.mg). Control activity was 540 (f56) pmol/(min.mg). Table 111. Fold Increase of Total P450 and Specific Isoforms of P450 Immunoquantitated by Western Blot Technique in Microsomal Preparations of Rats Treated with Different Inducers* inducer
total P450b
acetone pyrazole Arochlor 1254 6-NF isosafrole 3-MC
1.34 (f0.19) 0.86 (f0.14) 2.25 (f0.16) 1.87 (f0.06) 1.78 (f0.40) 2.20 (fO.10)
P450 1A' 0.95 (f0.03) 1.6 (f0.2) 5.8 (f0.3) 9.8 (f0.5) 10.4 (f1.5) 13.2 (f1.3)
P450 2Eld 1.65 (fO.l) 1.32 (f0.03) 0.52 (f0.04) 0.56 (f0.02) 0.42 (f0.02) 0.23 (f0.03)
a Values are reported as the mean ( M D ) of at least 3 rata. Total P450 determined by differential spectrophotometry (mean of 4 samples). Sum of P450 1Al + P450 1A2, relatively to the same amount of microsomal proteins (mean of 3 samples). Expressed relative to the same amount of proteins (mean of 2 samples).
days of culture, the 6-hydroxylation activity decreased by 2-fold, from 320 f 36 to 160 f 12 (measured at t = 4 h) pmoV24 hidish ( n = 4 experiments). This figure emphasized the effect of 3-MC treatment on this monooxygenase activity known to be supported by P450 2E1. Pyrazole (25)and ethanol appeared to be weak inducers of P450 2E1, like MezSO (23),in rat hepatocyte cultures. However, as the values of the 6-hydroxylation activity after such treatments were comparable to those of t = 4 h, these agents should be considered as protecting P450 2E1 from degradation more than inducing it. Therefore, this experiment clearly confirmed results obtained with human hepatocytes. In order to show that these observations were not pitfalls due to the hepatocyte model, similar experiments of induction were carried out in-vivo. In-Vivo Treatment of Rats. Rats were treated with two families of inducers: acetone or pyrazole, known to induce P450 2E1 (6, 261, and Arochlor 1254, 0-NF, isosafrole, and 3-MC, known to induce the P450 1A family (1). Table I1shows that acetone and pyrazole were weaker inducers of chlorzoxazone 6-hydroxylationthan isosafrole and 3-MC [acetone vs 3-MC, p < 0.1; pyrazole vs 3-MC, p < 0.001; pyrazole vs isosafole, p < 0.001; acetone vs isosafole, p < 0.2 (not significant)]. These latter compounds were strong inducers of two monooxygenase activities supported by P450 1AlllA2 enzymes, especially EROD involving preferentially P450 1Al (27). In order to verify that polyaromatic compounds did not modify the expression of P450 2E1, this enzyme was immunoquantitated by Western blot with anti-rat P450 2E1 antibody (Table 111). Unexpectedly, P450 2E1 decreased vs controls when rats were treated by Arochlor 1254,&NF, isosafrole, or 3-MC. On the other hand, the two enzymes of P450 1A family (lAl/lA2) increased
B7 5
T
2503
~,,w,,l
Control +a-NF
3-MC
3-MC+a-NF
J-MC+MqSO
Figure 2. Effect of different inhibitors on chlorzoxazone 6-hydroxylation measured in microsomal samples of control rata and 3-MC treated rata. Microsomal samples, 0.4 mg of protein, were incubated with 0.4 mM chlorzoxazone and 1 mM NADPH as described in the Materials and Methods section. The inhibitors were added at 50 pM and 28 mM concentrations, respectively, for a-naphthoflavone (a-NF) and dimethyl sulfoxide (Me2SO). Results are expressed as means standard deviation of three samples.
*
Table IV. Chlorzoxazone 6-Hydroxylation Activity of Microsomes from Yeasts ExDressing Human P480s* yeast strains
total P450b
chlorzoxazone 6-hydroxylationc
lAl-W(R) lAB-W(R) 2C9-W(R) 3A4-W(R) 2E1-PC-12 cellse
11.4 29.8 7.6 226.4 2.5
28.4 0.6 0.48 13.9 62
turnover no.d 2.50
0.02 0.06
0.06 25
a Control yeast had low activity [5.6 pmol/(min.mg)l which was substracted for P450 lAl,lA2,2C9, and 3A4. W(R) is a S. cereuisioe strain that overexpresseayeast NADPH-P450 reductase when grown with galactose as a carbon source. pmol/mg of protein. pmol/ (minsmg of protein). m i d . e According to ref 28.
significantly after such treatments while acetone and pyrazole treatments did not significantly modify the P450 1A content of microsomes. Figure 2 presents the effect of two inhibitors, namely, MezSO and CY-NF,on chlorzoxazone 6-hydroxylation determined in microsomal samples from control rats or rats treated with 3-MC. The inhibitor a-NFdid not modify this monooxygenase in control samples, while its effect was dramatic in 3-MC treated samples: 77% activity was inhibited. The addition of MezSO to the same microsomal preparations inhibited 39 % of chlorzoxazone 6-hydroxylation activity. Therefore, these results suggest that chlorzoxazone 6-hydroxylase activity measured in 3-MCtreated samples is mediated by two P450 families. At this step of work, the nature of the P4501A isoform involved in the metabolism of chlorzoxazone in addition to P450 2E1 could not be precisely ascertained. This goal was reached by using yeasts expressing different human P450s. Metabolism from Yeast Cells Expressing Human P450 lAl,lA2,2C9,and 3A4. Incubation of chlorzoxazone with microsomal preparations of yeast cells that were geneticallyengineered for stable expression of human P450 1A1, P450 1A2, P450 2C9, and P450 3A4 resulted in formation of 6-hydroxylated metabolite, as shown in Table IV. For the sake of comparison, data obtained with mammalian cells genetically modified for P450 2E1 expression (28)were included in Table IV. This table clearly demonstrates that P450 1Al is involved in the 6-hydroxylation reaction of chlorzoxazone.
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Carriere et al.
-l
0 00
0 05
0 10
01s
d 20
(25
d 30
l/iChlorzoxazonel (pM' 1
Figure 3. 6-Hydroxylation of chlorzoxazone by P450 1Al expressed in yeast. Representation was as a reciprocal plot of v [pmol/ (min-mgof protein)] againstchlorzoxazoneconcentrations (pM-l). Kinetic parmeters were determined: V, = 40 pmol/ (min-mgofprotein);K , = 217pM. Microsomalpreparationsfrom yeasts expressingP4501Al were used 2 mg of microsomal protein was incubated for 60 min with concentrations of chlorzoxazone ranging from 4 to 1200 pM. The regression line was calculated as follows: y = 0.025 + 5 . 4 3 ~(r = 0.98). The kinetic parameters of this enzymatic reaction were studied (Figure 3). It followed a monophasic MichaelisMenten kinetics, characterized by a K , of 217 pM and V , of 40 pmol/ (min-mg of protein).
Discussion The P450 superfamily is composed of families and subfamilies that are defined solely on the basis of their amino acid sequence similarities (1). They are characterized by a rather broad substrate specificity (3,291.Thus P450 1A1, which must be considered primarily an extrahepatic enzyme (I), is involved in the oxidation of polyaromatic hydrocarbons (30)while P4502E1 is involved in the oxidation metabolism of dialkylnitrosamines (31, 32) and many low molecular weight compound cancer suspects like halogenated solvents, benzene, styrene, halomethanes, and ethyl carbamate (33). As levels of P450 2E1 can vary considerably among individual humans (341, the use of drugs as noninvasive probe for determining the in-vivolevel of this enzyme woul be useful. This role could be played by chlorzoxazonewhich was shown to be oxidized only to the 6-hydroxy compound in human liver specifically by P450 2E1 (5). Although chlorzoxazone has been suggested by many authors to be an in-vivo probe to test P4502E1 activity, to our knowledge, no data are available to date. This observation raised questions about the suitability of chlorzoxazone as a noninvasive probe of human P450 2E1. Furthermore, in a study conducted in this laboratory to validate the usefulness of the conversion of chlorzoxazone to the 6-hydroxylated derivative in-vivo, preliminary data2 have shown that heavy smokers metabolized chlorzoxazone more efficiently than control subjects. Thus, this study was conducted to test the specificity of the chlorzoxazone 6-hydroxylation activity. The results of the work described here provided, for the first time, compelling evidence that P450 1Al is also involved in the 6-hydroxylation of chlorzoxazone in addition to P450 2E1. This evidence can be summarized as follows: (i) Treatment of rat or human hepatocyte cultures by 3-MC increased this monooxygenase activity by 3- and 3.6-fold, respectively. Ethanol, pyrazole, and MezSO D. Lucas, unpublished results. D. Lucas, unpublished results.
treatments also stimulated this activity. This latter result was expected because these three compounds are known to induce P450 2E1 in hepatocyte cultures (23,26,35,36). When cells were treated by 3-MC plus ethanol, the increase was more dramatic than with 3-MC alone, in rat as well as in human hepatocytes. 3-MC was shown to induce P450 1A2 (35) and both P450 1Al and P450 1A2 in cultured human hepatocytes (37). Furthermore, as ethanol itself was a strong inducer of P450 1Al and P450 1A2 in pure cultures of rat hepatocytes (36),it is not surprising that simultaneous treatment by 3-MC plus ethanol led to a dramatic increase of chlorzoxazone 6-hydroxylation. (ii) In-vivo treatments of rats by four inducers of P450 1A enzymes stimulated chlorzoxazone 6-hydroxylation more than acetone or pyrazole, both known to induce P450 2E1. The increase of this activity was not supported by P4502E1because such treatments were shown to decrease the P4502E1 protein. It is well known from animal studies (38,391 that both P450 1Al and 1A2 are expressed in a coregulated fashion. With 3-MC, P450 1 A l was shown to be induced 3-fold more than P450 1A2 (39,401 while with isosafrole P450 1A2 was induced 2-3-fold more than P450 lAl(38). 8-Naphthoflavone induced preferentially P450 lAl(17). Furthermore, as the level of induction is variable according to inducers, the increase of chlorzoxazone 6-hydroxylation activity could not attributed precisely to P4501Al or P4501A2. However, the contribution of P450 1A enzymes to the chlorzoxazone 6-hydroxylation was evaluated to about 75 5% in 3-MC treated samples vs about 35% for P450 2E1 because a-NF inhibited P450 1A and MezSO P450 2E1 monooxygenase activities in the same proportions, respectively. (iii) The use of microsomes from yeasts genetically engineered for cDNA expression of P450 1A1, 1A2,2C9, and 3A4 allowed us to answer to the question of the relative contributions of these P450s in the 6-hydroxylation of chlorzoxazone. Only P450 1Al is significantly involved in this catalytic activity. Its turnover number of 2.5 min-' was about 10-foldless than that of P450 2E1 expressed in mammalian cells (28). Our results raise questions about the suitability of chlorzoxazone as an in-vivo probe of hepatic P450 2E1 activity. As only P450 1A2 is expressed in human liver (41,42),the contribution of this enzyme to the chlorzoxazone 6-hydroxylation can be considered minor. As the level of CY2El is about %fold less than this of P450 3A4 (341, the major part of the 6-hydroxylation reaction is probably due to P450 2E1. Furthermore, as P450 2E1 has a much greater affinity for chlorzoxazone, at least in human hepatic microsomes, K , = 40pM (5),than pure P450 1A1, K , = 217 pM, the role of P450 1Al in 6-hydroxychlorzoxazone formation in-vivo a t the physiologic chlorzoxazone concentration of 30-60 pM3seems to be minor when compared to that of P450 2E1. However, in heavy smokers or subjects treated by drugs that are aryl hydrocarbonlike inducers ( 3 3 , the contribution of hepatic and extrahepatic P450 1Al cannot be excluded. In conclusion, chlorzoxazone should be used with precaution as an invivo tool for evaluation of P450 2E1 activity.
Acknowledgment. We are grateful to Dr. R. Peter (University of Erlangen-Ntimberg, Germany) for the gift of 6-hydroxychlorzoxazone. We acknowledge the skillful technical assistance of Ms. Y. Dreano and Mrs. N. Hourmant. We also wish to thank Ms. B. Marchix for careful typing of the manuscript.
Chlorzoxazone Is a Substrate of P450 2E1 and 1Al
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